Flashcards in Organic Chemistry- Separations and Purifications Deck (110):
What is one of the simplistways to separate out a desired product?
What is extraction?
The transfer of a dissolved compound (the desired product) from a starting solvent into a solvent in which the product is more soluable.
What is extraction based on?
The fundamental concept that like dissolves like. This principle tells us that a polar substance will disolve best in polar solvents, and a nonpolar substance will dissolve best in nonpolar solvents.
When performing extractions, it is important to make sure the two solvents are what?
What is immiscible?
They form two layers that do not mix, like water and oil
How do extractions work?
The two layers are temporarily mixed by shaking so that solute can pass from one solvent to the other.
What is the water layer called?
Aqueous phase (layer)
What is the nonpolar ether layer called?
Organic phase (layer)
How do you isolate the two phases once they have separated?
Use an equipment called a separatory funnel
Gravitational forces cause the denser layer to sink to the bottom of the funnel where it can then be removed by turning the top cock at the bottom.
What layer is most common to be on top?
Organic layer although the opposite can occur
The position of the layers is determined by what?
Their relative densities
Why are extractions done multiple times?
This is done in order to extract as much of the isobutyric acid from the ether layer as possible because it does not completely transfer with the first extraction . Multiple extraction with fresh water are more effective for obtaining the most product, rather than a single extraction with a larger volume of water.
Once the desired product has been isolated in the solvant, what is done?
We can obtain the product alone by evaporating the solvent, usually by using a rotary evaporator (rotovap)
What is a wash?
Perform the reverse of the extraction in order to removed unwanted impuries. A small amount of solute is used to extract and remove impurities rather than the compound of interest.
What does filtration isolate?
A solid from a liquid
How is filtration performed?
One pours a liquid-solid mixture onto a paper filter that allows only the solvent to pass through.
What is the result of filtration?
Left with the residue and filtrate
What is the residue?
What is the filtrate?
The flask full of liquid that passed through the filter
What is gravity filtration?
The solvent's own weight pulls it through the filter
When is gravity filtration used?
When the product of interest is in the filtrate
When is hot solvent generally used in filtration?
To keep the product dissolved in liquid
What is vaccum filtration?
The solvent is forced through the filter by a vaccum connected to the flask
When is vaccum filtration used?
When the solid is the desired product
Recrystallization is a method for what?
Further purifying crystals in solution
How is recrystallization performed?
we dissolve our product in a minimum amount of hot solvent and let it recrystallize as it cools.
How do you choose the solvent for recrystallization?
It should one in which the product is soluble only at high temperatures
Why is the solvent important in recrystalization?
When the solvent cools, only the desired product will recrystallize out of solution, excluding the impurities
When does distillation come in handy?
When the product itself is a liquid that is soluble in the solvent
What does distillation take advantage of?
Difference in boiling point to separate two liequids by evaporation and condensation
What happens in distillation?
The liquid with the lower boiling point will vaporize first, and the vapors will rise up the distillation column to condense in a water-cooled condenser. This condensation then drips down into a vessel
What is the end product called in distillation?
Why is the heating temperature kept low in distillation/
So that the liquid with the higher boiling point will not be able to boil and therefore will reamin liquid in the initial container.
What is the least complex version of distillation?
Simple distillation which proceeds as described earlier
simple distillation should only be used in what circumstances?
To separate liquids that boil below 150 degrees C and have at least a 25 degree C difference in boiling point
What apparatus is used in simple distillation?
Distilling flask containing the combined liquid solution
A distillation column consisting of a thermometer and a condenser
And a receiving flask to collect the distillate
When is vacuum distillation used?
Whenever we want to distill a liquid with a boiling point over 150 degrees C.
What does using a vacuum distillation do?
We lower the ambient pressure, thereby decreasing the temperatuer that the liquid must reacch in order to have sufficient vapor pressure to boil. This allows us to distill compound with higher boiling points at lower temperatures so that we do not have to worry about degrading the product
When is fractional distillation used?
To separate two liquids with similar boiling points (less than 25 degree C apart)
In fractional distillation what connects the distillation flask to the condensor?
A fractionation column
What is a fractionation column?
Is a column in which the surface area is increased by the inclusion of inert objects like glass beads or steel wool.
What happens in a fractionation column?
As the vapor rises up the column, it condenses on these beads or steel wool surfaces and refluxes back down until rising heat causes it to evaporate again, only to condense again higher in the column. Each time the condensate evaporates, the vapor consists of a higher proportion of the compound with the lower boiling point. By the time the top of the column is reached, only the desired product drips down to the receiving flask.
What is the general concept of chromatography?
The more similar a compound is to its surroundings (whether by polarity, charge, or other characteristics), the more it will stick to and move slowly through its surroundings.
What is the stationary phase in chromatography?
The process begins by placing the sample onto a solid medium called the stationary phase.
What is another name for stationary phase?
What is the mobile phase of chromatography?
Then run a liquid (or a gas ) through the stationary phase
What does the mobile phase do?
This will displace (elute) the sample and carry it through the stationary phase.
What is partitioning?
Depending on the characteristics of the substances in the sample and the polarity of the mobile phase, it will often adhere to the stationary phase with differing strengths, causing the different substances to migrate at different speeds.
Different compounds will have different _______ and will _____- at different rates?
Partitioning coefficients and will elute at different rates
What is the result of different partitioning coefficients?
Separation within the stationary phase, allowing us to isolate each substance individually.
What is the property most commonly used on the MCAT dealing with chromatography?
In column chromatography, what two things have a role in how quickly a coupound moves through the stationary phase?
Size and charge
Chromatography can even use strong interactions like what?
Chromatography is based on what?
The speed at which compounds move through media
In practice, how is chromatography measured?
Measure either how far each substance travels in a given amount of time (such as in TLC) or how long it takes to elute (as in column or gas chromatography)
What are the different types of chromatography?
Thin-layer and Paper chromatography
Gas Chromatogrphy (also called gas-liquid chromatography)
High-performance liquid Chromatography (HPLC)
What types of chromatography are very similar?
Thin layer and paper
What is the difference between thin-layer and paper chromatography?
Variance only in the medium used for the stationary phase
In thin-layer chromatography, what is used?
Silica gel or alumina adherent to an inert carrier sheet
In paper chromatography, what is used?
Paper composed of cellulose
How does thin-layer and paper work?
It starts with spotting, then the plate is developed. Then the solvent will creep up the plaste by capillary action, carrying the various compounds in the sample with it at varying rates. When the solvent front nears the top of the plate, the plate is removed from the chamber and allowed to dry.
What is spotting?
The sample that we want to separate is placed directly onto the adsorbent itself
Why is it called spotting?
Because we apply a small, well-defined spot of the sample directly onto the silica or paper plate.
What does a plate being developed mean?
It involves placing the adsorbent upright in a developing chamber, usually a beaker with a lid or a wide-mouthed jar. At the bottom of this jar is a shallow pool of solvent
What is the shallow pool or solvent called in paper or thin layer chromatography?
In thin-layer and paper chromatography, the spot of the sample must be what? Why?
Above the level of the solvent, or else they will dissolve into the pool of solvent rather than running up the plate
TLC is often done with _______ which is _______.
Silica gel which is polar and hyrdophilic
The mobile phase of the TLC is often what?
An organic solvent of weak to moderate polarity, so it doesn't bind well to the gel.
Because of the mobile phase in TLC what happens?
Nonpolar compounds dissolve in the organic solvent and move quickly as the solvent moves up the plate, whereas the more polar molecules stick to the gel. Thus the more nonpolar the sample is, the further up the plate it will move.
Reverse phase chromtography of TLC and paper is what and uses what? This results in what?
The exact opposite. The stationary phase used is nonpolar, so polar molecules move up the plate quickly, while nonpolar molecules stick more tightly to the stationary paper.
How can you see the spot on white paper in TLC and paper chromatography?
The developed plate can be placed under ultraviolet light, which will show any compounds that are ultraviolet-sensative. Alternatively, iodine, phosphomolybdic acid, or vanillin can be used to stain the spots, although this will destroy the compound such that they cannot be recovered.
When TLC is performed, compounds are identified using what?
retardation factor (Rf)
What is the retardation factor?
A relatively constant factor for a particular compound in a given solvent
How is the retardation factor calculated?
Rf= distance spot moved/distance solvent front moved
What can the R factor be used for? Why?
It can be used to identify unknown compound, because its value is relatively constant
What is preparative TLC?
As the large plate develops, the larger spot of samples splits into bands of individual compounds, which can then be scraped off and washed to yield pure compounds
What is the difference between TLC and column chromatography?
Column uses an entire column filled with silica or aluminum beads as an adsorbent, allowing for much greater separation.
Thin layer uses capillary action to move the solvent up the plate, whereas column chromatography uses gravity to move the solvent and compounds down the column
What is flash column chromatography? What is it used for?
One forces the solvent through the column using gas pressure. To speed up the process.
What can also be changed to elute the desired compound?
The solvent polarity can be changed
What are the different types of column Chromatography?
What happens after the solvent has separated in column chromatography?
The solvent drips out of the end of the column, and the different fractions that leave the column can be collected over time.
Each fraction in the column will contain different what?
Why is column chromatography useful in biochemistry?
It can be used to separate and collect macromolecules such as proteins or nucleic acids.
What happens in Ion-exchange chromotography?
The beads in the column are coated with charged substances so that they attract or bind compounds that have an opposite charge. For instance, a positively charged compound will attract and hold a negatively charged backbone of DNA or protein as it passes through the column, either increasing its retention time or retaining it completely. After all other compounds have moved through the column, a salt gradient is used to elute the charged molecules that have stuck to the column
What happens in size-exculsion chromatography?
The beads used in the column contain tiny pores of varying sizes. These pores allows small compounds to enter the beads, thus slowing them down. arge compounds can't fit into the pores, so they will move around them and travel through the column faster.
In size exclusion, Which size is slowed down
Smaller compounds are slowed down and retained longer
Why are the size of the pores varied in size exclusion?
So that molecules with different molecular weight can be fractionated.
What is a common approach in proton purification?
To use ion-exchange column followed by size exclusion column
What happens in Affinity chromotography?
A protein of interest is bound by creating a column with high affinity for that protein. This can be accomplished by coating beads with a receptor that bind the protein or a specific antibody to the protein, so the protein is retained in the column
What are common stationary phase molecules for affinity chromatography?
Nickel, which is used in separation of genertically engineered proteins with histidine tages
Antibodies or antigens
Enzymes substrate analogues, which mimic the natural substrate for an enzyme of interest.
What happens in Affinity once the protein is retained in the column?
It can be eluted by washing the column with a free receptor (or target or antibody), which will compete with the bead-bound receptor and ultimately free the protein from the column.
Eluents can also be created with what for Affinity Chromatography?
A varying pH or salinity level that disrupts the bonds between the ligand and the protein of interest
What is the drawback of the elution step in Affinity Chromatography?
The recovered substance can be bound to the eluent.
What is another name for gas chromatography?
vapor-phase chromatography (VPC)
What is the difference between gas and the other chromatographies?
The eluent is a gas intead of a liquid (usually helium or nitrogen)
What happens in VPC?
The adsorbent is a crushed metal or polymer inside a 30-foot column. This column is coiled and kept inside an over to control its temperature. The mixture is then injected into the column and vaporized. The gaseous compounds travel through the column at different rates because they adhere to the adsorbent in the column to different degrees and will separate in space by the time they reach the end of the column.
The injected compounds must be _______ in VPC
What is volatile?
Low melting-point, sublimable solids or vaporizable liquids
How are compounds registered in VPC?
By a detector, which records them as a peak on a chart
What is a common procedure involving VPC?
Separate molecules using VPC and then inject the pure molecules into a mass specrometer for molecular weight determination
What does mass spectroscopy involve?
The ionization and fragmentation of compounds; these fragments are then run through a magnetic field, which separates them by mass-charge ratio.
What can be determined after mass spectroscopy?
The total molecular weight or the relative concentrations of the different fragments can be calculated and compared against reference values to identify the compound
What was HPLC initally called?
High-pressure liquid chromatography
In HPLC, what is the eluent?
Where does the eluent travel in HPLC?
A column of a defined composition
In HPLC, there are a variety of _____ that can be chosen depending on ____ and the _____.
Variety of stationary phases
Depending on the target molecule and the quantity of material that needs to be purified.
In the past of HPLC, very high _____ was used.
How does HPLC work?
A small sample is injected into the column, and separation occurs as it flows through. the compounds pass through a detector and are collected as the solvent flows out of the end of the apparatus.
The interface for HPLC is similar to what? why?
That used for GC because the entire process is computerized, but uses liquid under pressure instead of gas.